Blockade of CD80/CD86-CD28 co-stimulation augments the inhibitory function of peptide antigen-specific regulatory T cells.

Mixed lymphocyte culture under the blockade of CD80/CD86-CD28 co-stimulation induces anergic (completely hyporesponsive) T cells with immune suppressive function (inducible suppressing T cells: iTS cells). Previously, iTS cell therapy has demonstrated outstanding benefits in clinical trials for organ transplantation. Here, we examined whether peptide antigen-specific iTS cells are inducible. DO 11.10 iTS cells were obtained from splenocytes of BALB/c DO 11.10 mice by stimulation with OVA peptide and antagonistic anti-CD80/CD86 mAbs. When DO 11.10 iTS or Foxp3- DO 11.10 iTS cells were stimulated with OVA, these cells produced IL-13, but not IL-4. DO 11.10 iTS cells decreased IL-4 and increased IL-13 production from OVA-stimulated naïve DO 11.10 splenocytes. When Foxp3+ DO 11.10 iTS cells were prepared, these cells significantly inhibited the production of IL-4 and IL-13 compared with freshly isolated Foxp3+ DO 11.10 T cells. Moreover, an increase in the population expressing OX40, ICOS, and 4-1BB suggested activation of Foxp3+ DO 11.10 iTS cells. Thus, blockade of CD80/CD86-CD28 co-stimulation during peptide antigen stimulation augments the inhibitory function of Foxp3+ regulatory T cells, and does not induce anergic Foxp3- conventional T cells. Peptide-specific Foxp3+ regulatory iTS cells could be useful for the treatment of allergic and autoimmune diseases without adverse effects.

CD8+ T cell-mediated rejection of allogenic human-induced pluripotent stem cell-derived cardiomyocyte sheets in human PBMC-transferred NOG MHC double knockout mice.

BACKGROUND: Transplantation of human-induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) has emerged as a promising therapy to treat end-stage heart failure. However, the immunogenicity of hiPS-CMs in transplanted patients has not been fully elucidated. Thus, in vivo models are required to estimate immune responses against hiPS-CMs in transplant recipients. METHODS: We transferred human peripheral blood mononuclear cells (hPBMCs) into NOD/Shi-scid IL-2rgnull (NOG) MHC class I/II double knockout (NOG-ΔMHC) mice, which were reported to accept hPBMCs without xenogeneic-graft-versus-host disease (xeno-GVHD). Then, hiPS-CM sheets generated from the hiPS cell line 201B7 harboring a luciferase transgene were transplanted into the subcutaneous space of NOG-ΔMHC mice. Graft survival was monitored by bioluminescent images using a Xenogen In Vivo Imaging System. RESULTS: The human immune cells were engrafted for more than 3 months in NOG-ΔMHC mice without lethal xeno-GVHD. The hiPS-CMs expressed a moderate level of human leukocyte antigen (HLA)-class I, but not HLA-class II, molecules even after interferon-gamma (IFN-γ) stimulation. Consistently, the allogenic IFN-γ-treated hiPS-CMs induced weak CD8+ but not CD4+ T cell responses in vitro. hiPS-CM sheets disappeared approximately 17 to 24 days after transplantation in hPBMC-transferred NOG-ΔMHC mice, and CD8+ T cell depletion significantly prolonged graft survival, similar to what was observed following tacrolimus treatment. CONCLUSIONS: hiPS-CMs are less immunogenic in vitro but induce sufficient CD8+ T cell-mediated immune responses for graft rejection in vivo.

Staphylococcus aureus δ-toxin present on skin promotes the development of food allergy in a murine model.

Background: Patients with food allergy often suffer from atopic dermatitis, in which Staphylococcus aureus colonization is frequently observed. Staphylococcus aureus δ-toxin activates mast cells and promotes T helper 2 type skin inflammation in the tape-stripped murine skin. However, the physiological effects of δ-toxin present on the steady-state skin remain unknown. We aimed to investigate whether δ-toxin present on the steady-state skin impacts the development of food allergy. Material and methods: The non-tape-stripped skins of wild-type, KitW-sh/W-sh, or ST2-deficient mice were treated with ovalbumin (OVA) with or without δ-toxin before intragastric administration of OVA. The frequency of diarrhea, numbers of jejunum or skin mast cells, and serum levels of OVA-specific IgE were measured. Conventional dendritic cell 2 (cDC2) in skin and lymph nodes (LN) were analyzed. The cytokine levels in the skin tissues or culture supernatants of δ-toxin-stimulated murine keratinocytes were measured. Anti-IL-1α antibody-pretreated mice were analyzed. Results: Stimulation with δ-toxin induced the release of IL-1α, but not IL-33, in murine keratinocytes. Epicutaneous treatment with OVA and δ-toxin induced the local production of IL-1α. This treatment induced the translocation of OVA-loaded cDC2 from skin to draining LN and OVA-specific IgE production, independently of mast cells and ST2. This resulted in OVA-administered food allergic responses. In these models, pretreatment with anti-IL-1α antibody inhibited the cDC2 activation and OVA-specific IgE production, thereby dampening food allergic responses. Conclusion: Even without tape stripping, δ-toxin present on skin enhances epicutaneous sensitization to food allergen in an IL-1α-dependent manner, thereby promoting the development of food allergy.

Anti-CD80/86 antibodies inhibit inflammatory reaction and improve graft survival in a high-risk murine corneal transplantation rejection model.

We investigated the effects of anti-CD80/86 antibodies in a murine high-risk corneal transplantation rejection model. A mixed lymphocyte reaction (MLR) assay was conducted with anti-CD80/86 antibodies. Inflammatory cytokine levels in the culture supernatant were measured using an enzyme-linked immunosorbent assay. Interferon (IFN)-γ-producing CD4+ T cell frequencies in the MLR were assessed using flow cytometry. In vivo, high-risk corneal allograft survival and IFN-γ-producing CD4+ T cell frequencies in corneal grafts were assessed with intraperitoneal injection of anti-CD80/86 antibodies compared to phosphate-buffered saline (PBS). RNA-sequencing was performed on corneal grafts 2 weeks post-transplantation. Anti-CD80/86 antibodies significantly decreased T-cell proliferation, IFN-γ+-producing CD4+ T cell frequencies, and IFN-γ, interleukin (IL)-1ß, IL-2, IL-10, and tumor necrosis factor-α production in the MLR compared to PBS injection. Intraperitoneal injection of anti-CD80/86 antibodies significantly prolonged corneal graft survival and decreased IFN-γ+-producing CD4+ T cell frequencies compared to PBS injection. Gene set enrichment analysis showed that the gene sets mainly enriched in the control group were related to allograft rejection and inflammatory response compared to PBS injection. Anti-CD80/86 antibodies significantly prolonged corneal graft survival by inhibiting T-cell proliferation and inflammatory response.

Anti-Donor Regulatory T-Cell Therapy in Adult-to-Adult Living Donor Liver Transplantation: A Case Report.

We report on the case of a 50-year-old female patient with symptomatic polycystic liver disease who underwent living donor liver transplantation (LDLT) using right liver graft from her ABO-identical husband. To achieve operational tolerance, regulatory T-cell (T-reg)-based cell therapy was applied, following the protocol introduced by Todo et al. Briefly, donor lymphocytes were collected by leukapheresis 20 days before LDLT without any adverse events, and the cells were irradiated with a dose of 30 Gy and kept frozen. Lymphopheresis of the recipient was conducted in a similar manner 1 day before LDLT, and donor cells and recipient cells were cultured with anti-CD80/86 antibodies to induce the donor-specific T-reg. At 14 days of culture, the CD4+CD25+Foxp3+ cells had increased from 1.51% to 5.21%, and mixed lymphocyte reaction assay using an intracellular fluorescent dye carboxyfluorescein diacetate succinimidyl ester-labeling technique revealed donor-specific hyporesponsiveness of CD4-positive lymphocytes. On postoperative day (POD) 13 (14 days of culture), these cells were infused to the recipient intravenously without any adverse events. Initial immunosuppression consisted of tacrolimus, steroid and mycophenolate mofetil (MMF), and cyclophosphamide (40 mg/kg) administered on POD 5. Both the steroid and MMF were continued until 4 weeks after LDLT, and the patient was discharged on POD 30 with normal liver function. On POD 52, the patient developed acute cellular rejection and received appropriate reinforcement of immunosuppressive therapy and is currently doing well with normal liver function 30 months after LDLT with reduced anti-donor allo-activity. In summary, T-reg therapy was safely performed in adult LDLT, and we are following the patient carefully to determine whether she can achieve operational tolerance in the future.

Ex Vivo-Induced Bone Marrow-Derived Myeloid Suppressor Cells Prevent Corneal Allograft Rejection in Mice.

Purpose: To investigate the effects of ex vivo-induced bone marrow myeloid-derived suppressor cells (BM-MDSCs) on allogeneic immune responses in corneal transplantation. Methods: Bone marrow cells from C57BL/6J (B6) mice were cultured with IL-6 and GM-CSF for four days. The ex vivo induction of the BM-MDSCs was assessed using flow cytometry, inducible nitric oxide synthase (iNOS) mRNA expression using reverse transcription-quantitative polymerase chain reaction, and nitric oxide (NO) production in allogeneic stimulation. T-cell proliferation and regulatory T-cell (Treg) expansion were investigated on allogeneic stimulation in the presence of ex vivo-induced BM-MDSCs. IFN-γ, IL-2, IL-10, and TGF-ß1 protein levels were measured using enzyme-linked immunosorbent assays. After subconjunctival injection of ex vivo-induced BM-MDSCs, the migration of the BM-MDSCs into corneal grafts, allogeneic corneal graft survival, neovascularization, and lymphangiogenesis were assessed using flow cytometry, slit-lamp microscopy, and immunohistochemistry. Results: The combination of GM-CSF and IL-6 significantly induced BM-MDSCs with increased iNos mRNA expression. The ex vivo-induced BM-MDSCs promoted NO release in allogeneic stimulation in vitro. The ex vivo-induced BM-MDSCs inhibited T-cell proliferation and promoted Treg expansion. Decreased IFN-γ and increased IL-2, IL-10, and TGF-ß1 production was observed in coculture of ex vivo-induced BM-MDSCs. Injected ex vivo-induced BM-MDSCs were confirmed to migrate into the grafts. The injected BM-MDSCs also prolonged corneal graft survival and prevented angiogenesis and lymphangiogenesis. Conclusions: The ex vivo-induced BM-MDSCs have suppressive effects on allogeneic immune responses and prolong corneal allograft survival via the iNOS pathway, indicating that they may be a potential therapeutic tool for corneal transplantation.

Analysis of therapeutic potential of monocytic myeloid-derived suppressor cells in cardiac allotransplantation.

BACKGROUND: Myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs) are attractive immune cells to induce immune tolerance. To explore a strategy for improving the efficacy of MDSC therapies, we examined the impact of adoptive transfer of several types of MDSCs on graft rejection in a murine heart transplantation model. METHODS: We analyzed the effects of induced syngeneic and allogeneic bone marrow-derived MDSCs (BM-MDSCs) on graft survival and suppressive capacity. We also compared the ability of syngeneic monocytic MDSCs (Mo-MDSCs) and polymorphonuclear MDSCs (PMN-MDSCs) to inhibit graft rejection and investigated the suppression mechanisms. RESULTS: Both syngeneic and allogeneic donor- or allogeneic third-party-derived BM-MDSCs prolonged graft survival, although syngeneic BM-MDSCs inhibited anti-donor immune responses most effectively in vitro. Syngeneic Mo-MDSCs, rather than PMN-MDSCs, were responsible for immune suppression through downregulating inducible nitric oxide synthase (iNOS) and expanded naturally occurring thymic originated Treg (nTreg) in vitro. Adoptive transfer of Mo-MDSCs, but not PMN-MDSCs, prolonged graft survival and increased Treg infiltration into the graft heart. CONCLUSION: Recipient-derived Mo-MDSCs are most effective in prolonging graft survival via inhibiting T cell response and nTreg infiltration.

PU.1 and IRF8 Modulate Activation of NLRP3 Inflammasome via Regulating Its Expression in Human Macrophages.

NLRP3 inflammasomes play crucial roles in the initiation of host defense by converting pro-Caspase-1 to mature Caspase-1, which in turn processes immature IL-1ß and IL-18 into their biologically active forms. Although NLRP3 expression is restricted to monocytic lineages such as monocytes, macrophages, and dendritic cells, the mechanisms determining the lineage-specific expression of NLRP3 remain largely unknown. In this study, we investigated the transcription factors involved in cell-type-specific transcription of NLRP3. We found that a distal, rather than a proximal, promoter of human NLRP3 was predominantly used in the human monocytic cell lines and macrophages. Reporter analysis showed that an Ets/IRF composite element (EICE) at -309/-300 and an Ets motif at +5/+8 were critical for transcriptional activity of the distal promoter. Electrophoretic mobility shift assays and chromatin immunoprecipitation assays demonstrated that two transcription factors, PU.1 and IRF8, both of which play essential roles in development and gene expression of the monocytic lineage, were bound to the EICE site, whereas PU.1 alone was bound to the Ets site. Knockdown of PU.1 and/or IRF8 mediated by small interfering RNA downregulated expression of NLRP3 and related molecules and markedly diminished the LPS-induced release of IL-1ß in THP-1, suggesting that activity of the NLRP3 inflammasome was suppressed by knockdown of PU.1 and IRF8. Taken together, these results indicate that PU.1 and IRF8 are involved in the monocytic lineage-specific expression of NLRP3 by binding to regulatory elements within its promoter and that PU.1 and IRF8 are potential targets for regulating the activity of the NLRP3 inflammasome.

Anti-CD321 antibody immunotherapy protects liver against ischemia and reperfusion-induced injury.

The prognosis of the liver transplant patients was frequently deteriorated by ischemia and reperfusion injury (IRI) in the liver. Infiltration of inflammatory cells is reported to play critical roles in the pathogenesis of hepatic IRI. Although T lymphocytes, neutrophils and monocytes infiltrated into the liver underwent IRI, we found that neutrophil depletion significantly attenuated the injury and serum liver enzyme levels in a murine model. Interestingly, the expression of CD321/JAM-A/F11R, one of essential molecules for transmigration of circulating leukocytes into inflammatory tissues, was significantly augmented on hepatic sinusoid endothelium at 1 h after ischemia and maintained until 45 min after reperfusion. The intraportal administration of anti-CD321 monoclonal antibody (90G4) significantly inhibited the leukocytes infiltration after reperfusion and diminished the damage responses by hepatic IRI (serum liver enzymes, inflammatory cytokines and hepatocyte cell death). Taken together, presented results demonstrated that blockade of CD321 by 90G4 antibody significantly attenuated hepatic IRI accompanied with substantial inhibition of leukocytes infiltration, particularly inhibition of neutrophil infiltration in the early phase of reperfusion. Thus, our work offers a potent therapeutic target, CD321, for preventing liver IRI.

Notch signaling contributes to the establishment of sustained unresponsiveness to food allergens by oral immunotherapy.

BACKGROUND: Oral immunotherapy (OIT) aims to establish desensitization and sustained unresponsiveness (SU) in patients with food allergy by ingestion of gradually increasing doses of specific food allergens. However, little is known about the mechanisms by which OIT induces SU to specific allergens. OBJECTIVES: We investigated the role of Notch signaling, which controls cell fate decisions in many types of immune cells in the induction of SU by OIT treatment. METHODS: Two types of mouse models, ovalbumin-induced food allergy and OIT, were generated. To elucidate the role of Notch signaling in OIT-induced SU, mice were intraperitoneally injected with the Notch signaling inhibitor N-[(3,5-difluorophenyl)acetyl]-l-alanyl-2-phenylglycine-1,1-dimethylethyl ester during the OIT treatment period. RESULTS: Ovalbumin-sensitized mice were desensitized and also had SU induced by OIT treatment, whereas repeated challenges with ovalbumin caused the development of severe allergic reactions in ovalbumin-sensitized mice. Administration of N-[(3,5-difluorophenyl)acetyl]-l-alanyl-2-phenylglycine-1,1-dimethylethyl ester to mice during the OIT treatment period inhibited the establishment of SU to ovalbumin but did not affect the induction of desensitization. OIT induced a systemic expansion of IL-10-producing CD4+ T cells, including TH2 cells, and myeloid-derived suppressor cells (MDSCs), particularly the monocytic MDSC subpopulation. Inhibition of Notch signaling prevented the OIT-induced expansion of those cells. In vitro cultures of bone marrow cells showed that Notch signaling directly promoted the generation of monocytic MDSCs. In addition, the contribution of MDSCs to OIT-induced SU was confirmed by MDSC depletion with the anti-Gr1 antibody. CONCLUSION: Notch signaling contributes to the establishment of SU induced by OIT through systemic expansion of immunosuppressive cells, such as IL-10-producing CD4+ T cells and MDSCs.

A Clinical Trial With Adoptive Transfer of Ex Vivo-induced, Donor-specific Immune-regulatory Cells in Kidney Transplantation-A Second Report.

BACKGROUND: Although the outcome of kidney transplantation (KTx) has improved, various adverse effects of immunosuppressants and chronic rejection aggravate the long-term prognosis of patients. Therefore, the induction of immune tolerance may be an effective therapeutic strategy. METHODS: A clinical trial aiming at immune tolerance induction was conducted in kidney transplant recipients from HLA mismatched living donors by infusing autologous donor-specific regulatory T cells (Treg). To obtain Treg, recipient's peripheral blood mononuclear cells were cocultured with irradiated donor cells in the presence of anti-CD80/CD86 monoclonal antibody for 2 weeks. For preconditioning, splenectomy + cyclophosphamide (CP) was employed in the first series (group A; n = 9). In group B, splenectomy was substituted by preadministration of rituximab (group B; n = 3). In the latest cases, rituximab + rabbit antithymocyte globulin was administered instead of cyclophosphamide (group C; n = 4). Twelve days after KTx, the cultured cells were intravenously infused, and immunosuppressants were gradually tapered thereafter. RESULTS: Although mixed lymphocyte reaction was remarkably suppressed in a donor-specific fashion, 6 out of 9 patients from group A, 1 out of 3 from group B, and 1 out of 4 from group C developed acute rejection within 1 year after KTx. Complete cessation of immunosuppression was not achieved, and a small dose of immunosuppressants was continued. CONCLUSIONS: The adoptive transfer of autologous ex vivo-expanded Treg is 1 of the options to possibly induce alloimmune hyporesponsiveness. However, in the present study, further regimen optimization is still required and should be the focus of future investigations.

PU.1 plays a pivotal role in dendritic cell migration from the periphery to secondary lymphoid organs via regulating CCR7 expression.

C-C chemokine receptor type 7 (CCR7) is essential for migration of dendritic cells (DCs) to draining lymph nodes. PU.1/Spi1 is a transcription factor playing a critical role in the gene regulation of DCs. PU.1 knockdown decreased the expression of CCR7 in bone marrow-derived DCs and subsequently attenuated migration in vitro and in vivo. Reporter assays, EMSA, and chromatin immunoprecipitation assays revealed that PU.1 binds to the most proximal Ets motif of the Ccr7 promoter, which is involved in transcriptional activation. The CCR7 expression level, which was higher in the programmed cell death 1 ligand 2 (PD-L2)+ population than in the PD-L2- population and was markedly suppressed by TGF-ß treatment, coincided with the binding level of PU.1 to the Ccr7 promoter. The PU.1 binding level in CCR7high mesenteric lymph nodes DCs was higher than in other DC subtypes. The involvement of PU.1 in the expression of the CCR7 gene was also observed in human DCs. We conclude that PU.1 plays a pivotal role in DC migration by transactivating the CCR7 gene via the Ets motif in the promoter in both humans and mice.-Yashiro, T., Takeuchi, H., Nakamura, S., Tanabe, A., Hara, M., Uchida, K., Okumura, K., Kasakura, K., Nishiyama, C. PU.1 plays a pivotal role in dendritic cell migration from the periphery to secondary lymphoid organs via regulating CCR7 expression.

Leukocyte mono-immunoglobulin-like receptor 8 (LMIR8)/CLM-6 is an FcRγ-coupled receptor selectively expressed in mouse tissue plasmacytoid dendritic cells.

Plasmacytoid dendritic cells (pDCs) produce large amounts of type-I interferon (IFN) in response to viral infection or self nucleic acids. Leukocyte mono-immunoglobulin-like receptor 8 (LMIR8), also called CMRF-35-like molecule-6 (CLM-6), is a putative activating receptor among mouse LMIR/CLM/CD300 members; however, the expression and function of LMIR8 remain unclear. Here, we characterize mouse LMIR8 as a pDC receptor. Analysis of Flag-tagged LMIR8-transduced bone marrow (BM)-derived mast cells demonstrated that LMIR8 can transmit an activating signal by interacting with immunoreceptor tyrosine-based activating motif (ITAM)-containing FcRγ. Flow cytometric analysis using a specific antibody for LMIR8 showed that LMIR8 expression was restricted to mouse pDCs residing in BM, spleen, or lymph node. FcRγ deficiency dampened surface expression of LMIR8 in mouse pDCs. Notably, LMIR8 was detected only in pDCs, irrespective of TLR9 stimulation, suggesting that LMIR8 is a suitable marker for pDCs in mouse tissues; LMIR8 is weakly expressed in Flt3 ligand-induced BM-derived pDCs (BMpDCs). Crosslinking of transduced LMIR8 in BMpDCs with anti-LMIR8 antibody did not induce IFN-α production, but rather suppressed TLR9-mediated production of IFN-α. Taken together, these observations indicate that LMIR8 is an FcRγ-coupled receptor selectively expressed in mouse tissue pDCs, which might suppress pDC activation through the recognition of its ligands.

The CD300e molecule in mice is an immune-activating receptor.

CD300 molecules (CD300s) belong to paired activating and inhibitory receptor families, which mediate immune responses. Human CD300e (hCD300e) is expressed in monocytes and myeloid dendritic cells and transmits an immune-activating signal by interacting with DNAX-activating protein 12 (DAP12). However, the CD300e ortholog in mice (mCD300e) is poorly characterized. Here, we found that mCD300e is also an immune-activating receptor. We found that mCD300e engagement triggers cytokine production in mCD300e-transduced bone marrow-derived mast cells (BMMCs). Loss of DAP12 and another signaling protein, FcRγ, did not affect surface expression of transduced mCD300e, but abrogated mCD300e-mediated cytokine production in the BMMCs. Co-immunoprecipitation experiments revealed that mCD300e physically interacts with both FcRγ and DAP12, suggesting that mCD300e delivers an activating signal via these two proteins. Binding and reporter assays with the mCD300e extracellular domain identified sphingomyelin as a ligand of both mCD300e and hCD300e. Notably, the binding of sphingomyelin to mCD300e stimulated cytokine production in the transduced BMMCs in an FcRγ- and DAP12-dependent manner. Flow cytometric analysis with an mCD300e-specific Ab disclosed that mCD300e expression is highly restricted to CD115+Ly-6Clow/int peripheral blood monocytes, corresponding to CD14dim/+CD16+ human nonclassical and intermediate monocytes. Loss of FcRγ or DAP12 lowered the surface expression of endogenous mCD300e in the CD115+Ly-6Clow/int monocytes. Stimulation with sphingomyelin failed to activate the CD115+Ly-6Clow/int mouse monocytes, but induced hCD300e-mediated cytokine production in the CD14dimCD16+ human monocytes. Taken together, these observations indicate that mCD300e recognizes sphingomyelin and thereby regulates nonclassical and intermediate monocyte functions through FcRγ and DAP12.

CD155-Transducing Signaling through TIGIT Plays an Important Role in Transmission of Tolerant State and Suppression Capacity.

The precise mechanism of how the regulatory T cell population elicits and maintains tolerant state in activated T cells is poorly understood. To address this issue, we established an in vitro coculture system using mouse T cells and showed that tolerant state is serially passed from preinduced-tolerant T cells into new TCR-stimulated T cells across generations in a dendritic cell-independent manner. In this successive induction process of tolerant state, TIGIT was found to play an important role: TIGIT expression on induced-tolerant T cells was promoted in stimulated T cells cocultured with the tolerant cells. In addition, these stimulated T cells in the coculture also expressed high B lymphocyte-induced maturation protein 1 accompanied by IL-2 suppression. Because CD155, a partner of TIGIT, is known to transduce signaling inside by trans-interaction with its ligands, these phenotypical changes in TCR-stimulated naive T cells were reproduced when naive T cells were double cross-linked by CD3 and CD155. These results indicate that TIGIT enhanced on tolerant T cells may function as a ligand of its paired receptor CD155 to transduce signaling into its expressing naive T cells to accelerate new TIGIT expressions as well as IL-2 suppression via B lymphocyte-induced maturation protein 1 enhancement. In consideration of these results, we propose a novel process in which tolerant state in T cell population is maintained by successive generation of new tolerant T cells from naive T cells as one of the regulating mechanisms in immune responses.